Adhesion of L1210 cells to sulfonated styrene copolymer surfaces: imaging of F-actin AND alpha-actinin.

The static adhesion of living L1210 cells to sulfonated copolymer surfaces of different sulfonic group content and the actin cytoskeleton organization in the adhering cells were studied. The strength of the cell-substratum interaction was estimated by determining the relative number of cells remaining adherent despite experiencing a shearing force equal to 1.25 x 10(-11) N caused by the laminar flow of the medium. The cell-substratum interaction took place in a medium with or without serum. The distribution of F-actin and alpha-actinin in the adhering cells was determined in sequences of fluorescent images of cell optical slices with the use of a computer method of cell image analysis. It was shown that the surface sulfonic groups affect not only the rate and strength of cell-substratum adhesion but also the F-actin and alpha-actinin distribution (in the cell regions near the substratum surface) in cells adhering in the medium containing serum. These proteins, concentrated in the tips of microvilli, were observed as dots. The distinctness (discernibleness) and sizes of these dots depend on the surface content of sulfonic groups. F-actin is located at the periphery of the cells in cells adhering in the medium without serum and alpha-actinin is concentrated in small dots at the periphery and in the central part of the cells.

Cell adhesion to polymeric surfaces: experimental study and simple theoretical approach.

In a medium without serum, the initial adhesion of L1210 cells to nonsulfonated and sulfonated polymer surfaces was investigated. In the case of sulfonated polymer surfaces, the relative number of adhering cells strongly increases with an increase of the interfacial surface tension; that is, adhesion strongly depends on the surface density of sulfonic groups. However, in the case of nonsulfonated polymer surfaces, the relative number of adhering cells is high and independent of the interfacial surface tension. To extend the basic knowledge of these phenomena, a semi-empirical quantum chemical computational study was undertaken. Simple probe molecules were chosen that mimic the chemical properties of functional groups present on polymeric surfaces. The energies of interaction between these molecules and ones representing the midchain polypeptide building blocks were calculated. To discuss the steric effects involved in similar interactions on real surfaces, a simple model of polymeric surfaces was proposed. Also the interactions among such surfaces and the short hydrated polypeptide chain were studied at the molecular mechanics level of theory. The derived intermolecular energy parameter was found to change in parallel to the number of adhered cells within the two groups of substrata under study: nonsulfonated and sulfonated. The computational results suggest the possible existence of differently arranged cell membrane protein centers responsible for docking to these two types of surfaces.

Adhesion of L1210 cells to modified styrene copolymer surfaces in the presence of serum.

The static adhesion of living L1210 cells (in serum-containing medium) to the surface of (styrene/methylmethacrylate) copolymers differing in styrene content (from 5% to 50% of styrene units) was investigated. The examination of wettability of the copolymer surfaces showed that the contact angle of water on the hydrophobic surfaces is an increasing linear function of styrene content in the copolymer. Cell adhesion to the unwettable surfaces is low (within 2-4%). A novel method of modification of the styrene copolymer surfaces was used to render these surfaces suitable for cell attachment. The modification consists of sulfonation of the surfaces with sulfur trioxide at the gas/solid interface. The contact angle of sulfonated copolymer surfaces is a decreasing linear function of styrene content in the copolymer. The contact angle decreases due to the increased number of highly hydrophilic sulfonic groups bonded to styrene. By acetylation of the sulfonated surfaces it was shown that cell adhesion to acetylated surfaces is not diminished and is at the same level as cell adhesion to sulfonated copolymer surfaces. Thus, it can be concluded that sulfonation of copolymer surfaces does not form hydroxyl groups. Cell adhesion to substrata of high wettability stabilizes after 30s. The relative number of cells adhering to the sulfonated copolymer surfaces is a decreasing linear function of the contact angle. For the copolymer surfaces containing 50% of styrene units the contact angle decreases sevenfold, due to sulfonation, and the number of adhering cells increases 40-fold. The results obtained show that for cell-substratum adhesive interaction the presence of sulfonic groups at the substratum surface is important.

Effect of cell-substrate interaction time and shearing force on adhesion of L1210 cells to collagen and glass.

The early adhesive interaction of living cells with substrates was examined. L1210 cells were allowed to interact with collagen or glass in serum-containing medium (time of cell-substrate interaction, tint, under stationary conditions, was within 2-25 min) or in serum-free medium (tint was 5 s-15 min). The relative number A of cells adhering under stationary conditions, and remaining adherent to the substrate despite experiencing a shearing force F, was determined. The following was found for cells adherent to collagen and glass, both in the presence and absence of serum in the medium. The number A increases with the value of tint and tends to reach a plateau. The plateau value depends on adhesion conditions (presence or absence of serum). When adhesion occurred in serum-containing medium (F congruent to 0.1 X 10(-13)N), function A(tint) increased up to 15 min. The plateau values were in the ratio of 2:1 for cells adherent to collagen and glass, respectively. When adhesion took place in serum-free medium, the function A(tint) increased within the first 20 or 130s for cells adhering to glass or collagen, respectively. The value of A(tint) increased up to the effective interaction time, teff int, i.e. the time after which a plateau was reached at 100% adhesion. This meant that after tint greater than or equal to teff int all cells were in adhesion with glass and collagen despite the application of the greatest shearing force, F congruent to 2 X 10(-9)N. The values of teff int for cells adherent to collagen and glass were in the ratio of 6:1, respectively. The value of A decreases with the value of F for cells adherent to substrate in the absence of serum when tint less than teff int. The function A(F) for cells adherent to collagen and glass in the presence of serum, but not in the absence of serum, can be described by the equation: A = a/square root F + c, where a and c = constant greater than 0. The values of a were in the ratio of 2:1 for cells adherent to collagen and glass, respectively. The Brownian motion of cells interacting with both substrates in the absence of serum ceased. The times during which cell motion persisted for cells interacting with collagen and glass, respectively, were in the ratio of 2.5:1.

Adhesion and locomotion of L5222 cells on endothelium, collagen and glass.

Examination was made of the adhesive interaction of L5222 leukemia cells with endothelial cells, collagen and glass and of cell locomotion on endothelium and collagen. Leukaemia cells interacted with the substrate under stationary conditions. The fraction of cells adherent to the substrate was defined next, using the given shearing force caused by the medium flowing through the measuring channel. The relative number of adherent cells, A (related to the number of cells after sedimentation), remaining on the given surface despite the detaching action of shearing force, F, was determined. The range of F values applied was 0.1 to 30 (x 10(-13)) N. It was found that the relation A(F) is a decreasing function for all the substrates examined and takes on values in the relation 1:2:6 for cells adherent to collagen, glass and endothelium, respectively. The critical value of F, at which values of A are maintained at a constant level close to zero, was 0.5, 1 and 3 (x 10(12)) N, respectively, for cells adherent to these substrates. The function A(F) for L5222 cells adherent to endothelial cells and to glass can be described well by the formula: A = a/square root F (where a = constant greater than 0). Studies of L5222 cell locomotion on an endothelial cell layer and on collagen revealed that the pattern of locomotion, variations in locomotion velocity and the mean values of cell displacement (5.8 and 5.0 micrometers, respectively) are similar for both substrates.

Influence of flow velocity and cell concentration on dynamic adhesion of erythrocytes to glass surface.

Comparative studies were carried out on dynamic adhesion of 51Cr-labelled erythrocytes to the surface of glass beads in the presence of serum in the medium (50 microng of protein/ml) and in protein-free medium. The influence of cell concentration (within the range 4 X 10(5) to 8 X 10(6)/ml) and of cellular flow velocity (within the range 1.5-0.4 cm/min) on the value of adhesion was investigated. It was found that when serum was present in the medium, the decisive influence on erythrocyte adhesion was exerted by the velocity with which the cells pass though the glass bead layer. Cell concentration under these conditions has only a very slight effect. When the medium does not contain serum, erythrocyte adhesion to the bead layer seems to depend on both cell concentration and flow velocity. Preliminary data were obtained concerning the release of 51Cr from the bead layer after erythrocyte adhesion.

Dynamic adhesion of erythrocytes to glass. Comparative study of the influence of serum in low concentrations.

Adhesion of 51Cr-labeled rat erythrocytes was examined in a layer of glass beads in a dynamic system. The kinetics of erythrocyte retention in the bead layer was found to change with time. Adhesion decreased with time at all the studied rates of suspension flow through the layer (within the range 1-5--0-4 cm/min), both in presence and absence of serum in the medium. Serum in low concentrations of 20--200 microgram of protein per ml considerably inhibited dynamic adhesion of erythrocytes to glass. The higher the serum concentration in a medium (at a definite flow velocity), the quicker the decrease of adhesion with time, and the stronger the dependence of the kinetics of erythrocyte retention on the flow velocity of the cells. Mean adhesion of erythrocytes after 17-min perfusion of the layer (at 200 microgram of serum protein per ml of medium) seems to be independent of the concentration of the cells in the suspension flowing into the layer within the range of studied concentrations, 2 x 10(6)--20 x 10(6) cells/ml. However, mean adhesion of erythrocytes deprived of serum seems to be dependent on the cell concentration.